The Experts below are selected from a list of 249 Experts worldwide ranked by ideXlab platform
David Stanley - One of the best experts on this subject based on the ideXlab platform.
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prostaglandins and other eIcosanoids in insects biosynthesis and biological actions
Frontiers in Physiology, 2019Co-Authors: David StanleyAbstract:This essay reviews the discoveries, synthesis and biological significance of prostaglandins (PGs) and other eIcosanoids in insect biology. It presents the most current – and growing – understanding of the insect mechanism of PG biosynthesis, provide an updated treatment of known insect phospholipase A2 (PLA2), and detail contemporary findings on the biological roles of PGs and other eIcosanoids in insect physiology, including reproduction, fluid secretion, hormone actions in fat body, immunity and eIcosanoid signaling and cross-talk in immunity. It completes the essay with a prospectus meant to illuminate research opportunities for interested readers. In more detail, cellular and secretory types of PLA2, similar to those known on the biomedical background, have been identified in insects and their roles in eIcosanoid biosynthesis documented. It highlights recent findings showing that eIcosanoid biosynthetic pathway in insects is not identical to the solidly established biomedical picture. The relatively low concentrations of arachidonic acid (AA) present in insect phospholipids (< 0.1% in some species) indicate that PLA2 may hydrolyze linoleic acid (LA) as a precursor of eIcosanoid biosynthesis. The free LA is desaturated and elongated into AA. Unlike vertebrates, AA is not oxidized by cyclooxygenase, but by a specific peroxidase called peroxinectin to produce PGH2, which is then isomerized into cell-specific PGs. In particular, PGE2 synthase recently identified converts PGH2 into PGE2. In the cross-talks with other immune mediators, eIcosanoids act as downstream signals because any inhibition of eIcosanoid signaling leads to significant immunosuppression. Because host immunosuppression favors pathogens and parasitoids, some entomopathogens evolved a PLA2 inhibitory strategy activity to express their virulence.
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EIcosanoid-mediated immunity in insects
Developmental and Comparative Immunology, 2017Co-Authors: Shabbir Ahmed, David Stanley, Chunju AnAbstract:Abstract EIcosanoid is a collective term for oxygenated metabolites of C20 polyunsaturated fatty acids. As seen in mammals, eIcosanoids play crucial roles in mediating various physiological processes, including immune responses, in insects. Upon microbial pathogen infection, non-self recognition signals are propagated to nearly immune effectors such as hemocytes and fat body using various immune mediators, in which eIcosanoid signals act as the ultimate downstream mediator. The chemical diversity of eIcosanoids may operate to mediate various immune responses. Some entomopathogenic bacteria suppress eIcosanoid biosynthesis, which inhibits host insect immunity and promotes their pathogenicity. This review introduces immune responses mediated by various eIcosanoids. Then it explains the cross-talks of eIcosanoids with other immune mediators including cytokines, biogenic monoamines, and nitric oxide to clarify the complexity of insect immune mediation. Finally, we highlight the biological significance of eIcosanoids by demonstrating bacterial pathogenicity inhibiting a key enzyme – phospholipase A 2 – in eIcosanoid biosynthesis using their secondary metabolites to defend host insect immune attack.
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nitric oxide mediates insect cellular immunity via phospholipase a2 activation
Journal of Innate Immunity, 2017Co-Authors: David StanleyAbstract:After infection or invasion is recognized, biochemical mediators act in signaling insect immune functions. These include biogenic amines, insect cytokines, eIcosanoids, and nitric oxide (NO). Treating insects or isolated hemocyte populations with different mediators often leads to similar results. Separate treatments with an insect cytokine, 2 biogenic amines, and an eIcosanoid lead to a single result, hemocyte spreading, understood in terms of intracellular cross-talk among these signaling systems. This study focuses on the cross-talk between NO and eIcosanoid signaling in our model insect, Spodoptera exigua. Bacterial injection increased NO concentrations in the larval hemocytes and fat body, and RNA interference (RNAi) of the S. exigua NO synthase (NOS) gene suppressed NO concentrations. RNAi treatment also led to a significant reduction in hemocyte nodulation following bacterial injection. Similar RNAi treatments led to significantly reduced PLA2 activities in the hemocytes and fat body compared to control larvae. Injection of L-NAME also prevented the induction of PLA2 activity following bacterial challenge. An injected NO donor, S-nitroso-N-acetyl-DL-penicillamine, increased PLA2 activity in a dose-dependent manner. However, eIcosanoids did not influence NO concentrations in immune-challenged larvae. We infer that NO and eIcosanoid signaling operate via cross-talk mechanisms in which the elevated NO concentrations activate PLA2 and eIcosanoid biosynthesis, which finally mediates various immune responses.
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the influence of chronic eIcosanoid biosynthesis inhibition on life history of the greater waxmoth galleria mellonella and its ectoparasitoid bracon hebetor
Journal of Insect Physiology, 2011Co-Authors: Ender Büyükgüzel, Hasan Tunaz, David Stanley, Kemal BuyukguzelAbstract:EIcosanoids are oxygenated metabolites of three C20 polyunsaturated fatty acids, mainly arachidonic acid (AA; 20:4n-6), but also 20:3n-6 and 20:5n-3. Aside from their importance in biomedicine, eIcosanoids act in invertebrate biology. Prostaglandins (PGs) influence salt and water transport physiology in insect rectal epithelia and in Malpighian tubules. PGs also influence a few insect behaviors, including releasing oviposition behavior and behavioral fever. EIcosanoids act in ovarian development and in insect immunity. Because eIcosanoids act in several areas of insect biology, we posed the hypothesis that chronic inhibition of eIcosanoid biosynthesis, in the absence of microbial challenge, can influence insect life table parameters, including developmental time, survival, adult longevity and parasitoid fecundity. Here we report that inhibiting eIcosanoid biosynthesis throughout the larval life exerted minor influences on some life table parameters of the greater wax moth, Galleria mellonella and its ectoparasitoid, Bracon hebetor, however, the inhibitors strongly reduced the production and hatchability of the parasitoids' eggs. The significance of the work relates to the potentials of understanding and targeting eIcosanoid systems as a platform for developing new technologies of insect pest management. As seen here, the impact of targeting eIcosanoid systems is seen in crucial moments of insect life histories, such as reproduction or immune challenge rather than in overall larval development.
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An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits hemocytic phospholipase A2 (PLA2) in tobacco hornworms Manduca sexta
Journal of Invertebrate Pathology, 2004Co-Authors: Youngjin Park, Hasan Tunaz, David StanleyAbstract:Abstract The entomopathogenic bacterium, Xenorhabdus nematophila , induces immunodepression in target insects and finally leads to lethal septicemia of the infected hosts. A hypothesis has been raised that the bacteria inhibit eIcosanoid-biosynthesis pathway to interrupt immune signaling of the infected hosts. Here, we show direct evidence that X. nematophila inhibits the activity of phospholipase A2 (PLA2), the initial step in the eIcosanoid-biosynthesis pathway. Inhibition of PLA2 was dependent on both incubation time with X. nematophila and the bacterial concentration in in vitro PLA2 preparations of Manduca sexta hemocytes. While living bacteria inhibited PLA2 activity, heat-killed X. nematophila rather increased PLA2 activity. X. nematophila secreted PLA2 inhibitor(s) which were detected in the organic, but not aqueous, extract of the bacterial culture medium. The PLA2 inhibitory activity of the organic extract was lost after heat treatment. These results clearly indicate that X. nematophila inhibits PLA2 activity, and thereby inhibits eIcosanoid biosynthesis which leads to immunodepression of the infected hosts.
Jon S Miller - One of the best experts on this subject based on the ideXlab platform.
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eIcosanoids exploiting insect immunity to improve biological control programs
Insects, 2012Co-Authors: David W. Stanley, Eric J. Haas, Jon S MillerAbstract:Insects, like all invertebrates, express robust innate, but not adaptive, immune reactions to infection and invasion. Insect immunity is usually resolved into three major components. The integument serves as a physical barrier to infections. Within the hemocoel, the circulating hemocytes are the temporal first line of defense, responsible for clearing the majority of infecting bacterial cells from circulation. Specific cellular defenses include phagocytosis, microaggregation of hemocytes with adhering bacteria, nodulation and encapsulation. Infections also stimulate the humoral component of immunity, which involves the induced expression of genes encoding antimicrobial peptides and activation of prophenoloxidase. These peptides appear in the hemolymph of challenged insects 6–12 hours after the challenge. Prostaglandins and other eIcosanoids are crucial mediators of innate immune responses. EIcosanoid biosynthesis is stimulated by infection in insects. Inhibition of eIcosanoid biosynthesis lethally renders experimental insects unable to clear bacterial infection from hemolymph. EIcosanoids mediate specific cell actions, including phagocytosis, microaggregation, nodulation, hemocyte migration, hemocyte spreading and the release of prophenoloxidase from oenocytoids. Some invaders have evolved mechanisms to suppress insect immunity; a few of them suppress immunity by targeting the first step in the eIcosanoid biosynthesis pathways, the enzyme phospholipase A2. We proposed research designed to cripple insect immunity as a technology to improve biological control of insects. We used dsRNA to silence insect genes encoding phospholipase A2, and thereby inhibited the nodulation reaction to infection. The purpose of this article is to place our view of applying dsRNA technologies into the context of eIcosanoid actions in insect immunity. The long-term significance of research in this area lies in developing new pest management technologies to contribute to food security in a world with a rapidly growing human population.
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EIcosanoids mediate nodulation reactions to bacterial infections in larvae of the butterfly, Colias eurytheme.
Comparative Biochemistry and Physiology Part C: Pharmacology Toxicology and Endocrinology, 1999Co-Authors: David Stanley, Hasan Tunaz, Rico L Rana, A.r. Nor Aliza, Jon C Bedick, William Wyatt Hoback, Jon S MillerAbstract:Abstract Nodulation is the first, and qualitatively predominant, cellular defense reaction to bacterial infections in insects. Treating larvae of the butterfly Colias eurytheme with the eIcosanoid biosynthesis inhibitor dexamethasone, strongly impaired nodulation reactions to bacterial infections. The influence of dexamethasone was reversed by treating infected insects with arachidonic acid, an eIcosanoid precursor. An eIcosanoid biosynthesis system in C. eurytheme larvae is documented. Specifically, the presence of eIcosanoid-precursor polyunsaturated fatty acids in tissue phospholipids was determined, an intracellular phospholipase A2 that can release arachidonic acid from tissue phospholipids was recorded, and eIcosanoid biosynthesis, registered as conversion of exogenous radioactive 20:4n–6 into eIcosanoids, was observed. These findings support the hypothesis that eIcosanoids mediate cellular immune responses to bacterial infections in these butterfly larvae, and more broadly, in most, if not all, insects.
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EIcosanoids Mediate Nodulation Responses to Bacterial Infections in Larvae of the Silkmoth, Bombyx mori
Comparative Biochemistry and Physiology Part A: Physiology, 1997Co-Authors: David W. Stanley-samuelson, Venkat K Pedibhotla, Rico L Rana, Nor Aliza Abdul Rahim, William Wyatt Hoback, Jon S MillerAbstract:Abstract 1) Nodulation is the first, and qualitatively predominant, cellular defense reaction to bacterial infections in insects and other invertebrates; 2) treating silkworms, Bombyx mori, with the eIcosanoid biosynthesis inhibitor, dexamethasone, strongly reduced nodulation responses to bacterial infections; 3) the influence of dexamethasone was reversed by injecting the eIcosanoid-precursor polyunsaturated fatty acid, arachidonic acid (20:4n-6), into dexamethasone-treated, infected larvae; 4) the presence of an eIcosanoid biosynthesis system in silkworms was documented. Demonstrated elements include a digestive phospholipase A2, incorporation of exogenous 20:4n-6 into fat body phospholipids, the presence of 20:4n-6 in cellular phospholipids, a fat body intracellular phospholipase A2 that can hydrolyze 20:4n-6 from cellular phospolipids, and eIcosanoid biosynthetic enzymes; and 5) these findings support the hypothesis that eIcosanoids mediate cellular immune responses to bacterial infections in silkworms.
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EIcosanoids mediate microaggregation and nodulation responses to bacterial infections in black cutworms, Agrotis ipsilon, and true armyworms, Pseudaletia unipuncta
Journal of Insect Physiology, 1997Co-Authors: Russell A. Jurenka, Jon S Miller, Venkat K Pedibhotla, Rico L Rana, David W. Stanley-samuelsonAbstract:Nodulation is the first, and quantitatively predominant, cellular defense reaction to bacterial infection in insects and other invertebrates. Inhibition of eIcosanoid biosynthesis in true armyworms, Pseudaletia unipuncta, and black cutworms, Agrotis ipsilon, immediately prior to intrahemocoelic injections with heat-killed preparations of the bacterium, Serratia marcescens, severely impaired the nodulation response. Five eIcosanoid biosynthesis inhibitors, including dexamethasone (a phospholipase A2 inhibitor), indomethacin, ibuprofen (cyclooxygenase inhibitors), phenidone (dual lipoxygenase/cyclooxygenase inhibitor) and eicosatetraynoic acid (an arachidonic acid analog that inhibits all arachidonic acid metabolism) severely reduced nodulation in infected insects. The dexamethasone effects were reversed by treating true armyworms with arachidonic acid immediately after infection. In addition to these pharmacological findings, we demonstrate that an eIcosanoid biosynthesis system is present in these insects. Arachidonic acid is present in fat body phospholipids at about 0.4% of total phospholipid fatty acids. Fat body expressed a phospholipase A2 that can hydrolyze arachidonic acid from the sn-2 position of cellular phospholipids. Fat body preparations were competent to biosynthesize prostaglandins, of which PGE2 was the major product. These findings support the hypothesis that eIcosanoids mediate cellular immune reactions in insects.
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EIcosanoids mediate nodulation responses to bacterial infections in larvae of the tenebrionid beetle, Zophobas atratus
Journal of Insect Physiology, 1996Co-Authors: Jon S Miller, Ralph W. Howard, Tuan-duc Nguyen, A. Nguyen, R. M. T. Rosario, David W. Stanley-samuelsonAbstract:Nodulation is the temporally and quantitatively predominant cellular defense response to bacterial infection in insects and other invertebrates. Inhibition of eIcosanoid biosynthesis in larvae of the tenebrionid beetle, Zophobas atratus, immediately prior to intrahemocoelic injections with heat killed preparations of the bacterium, Serratia marcescens, strongly reduced the nodulation response. Separate treatments with specific inhibitors of phospholipase A2, cyclooxygenase, and lipoxygenase reduced nodulation, supporting the view that nodule formation is a complex process involving both cyclooxygenase and lipoxygenase products. The inhibitory effects of the phospholipase A2 inhibitor, dexamethasone, on nodulation were apparent 1 h after infection, and the effects increased, relative to controls, over 24 h. The dexamethasone effects were expressed in a dose-dependent manner, and they were reversed by treating bacteria injected insects with the eIcosanoid-precursor polyunsaturated fatty acid, arachidonic acid (C20:4n-6). Treatments with the saturated fatty acid, 16:0, which is not an eIcosanoid precursor, did not reverse the dexamethasone effects on nodulation. The insects contain low levels of three eIcosanoid precursor polyunsaturated fatty acids in six different tissues, and fat body preparations are competent to produce both cyclooxygenase and lipoxygenase products. These findings strongly support the identification of nodulation as a specific insect cellular defense mechanism that is mediated by eIcosanoids.
David W. Stanley-samuelson - One of the best experts on this subject based on the ideXlab platform.
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EIcosanoids Mediate Nodulation Responses to Bacterial Infections in Larvae of the Silkmoth, Bombyx mori
Comparative Biochemistry and Physiology Part A: Physiology, 1997Co-Authors: David W. Stanley-samuelson, Venkat K Pedibhotla, Rico L Rana, Nor Aliza Abdul Rahim, William Wyatt Hoback, Jon S MillerAbstract:Abstract 1) Nodulation is the first, and qualitatively predominant, cellular defense reaction to bacterial infections in insects and other invertebrates; 2) treating silkworms, Bombyx mori, with the eIcosanoid biosynthesis inhibitor, dexamethasone, strongly reduced nodulation responses to bacterial infections; 3) the influence of dexamethasone was reversed by injecting the eIcosanoid-precursor polyunsaturated fatty acid, arachidonic acid (20:4n-6), into dexamethasone-treated, infected larvae; 4) the presence of an eIcosanoid biosynthesis system in silkworms was documented. Demonstrated elements include a digestive phospholipase A2, incorporation of exogenous 20:4n-6 into fat body phospholipids, the presence of 20:4n-6 in cellular phospholipids, a fat body intracellular phospholipase A2 that can hydrolyze 20:4n-6 from cellular phospolipids, and eIcosanoid biosynthetic enzymes; and 5) these findings support the hypothesis that eIcosanoids mediate cellular immune responses to bacterial infections in silkworms.
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EIcosanoids mediate microaggregation and nodulation responses to bacterial infections in black cutworms, Agrotis ipsilon, and true armyworms, Pseudaletia unipuncta
Journal of Insect Physiology, 1997Co-Authors: Russell A. Jurenka, Jon S Miller, Venkat K Pedibhotla, Rico L Rana, David W. Stanley-samuelsonAbstract:Nodulation is the first, and quantitatively predominant, cellular defense reaction to bacterial infection in insects and other invertebrates. Inhibition of eIcosanoid biosynthesis in true armyworms, Pseudaletia unipuncta, and black cutworms, Agrotis ipsilon, immediately prior to intrahemocoelic injections with heat-killed preparations of the bacterium, Serratia marcescens, severely impaired the nodulation response. Five eIcosanoid biosynthesis inhibitors, including dexamethasone (a phospholipase A2 inhibitor), indomethacin, ibuprofen (cyclooxygenase inhibitors), phenidone (dual lipoxygenase/cyclooxygenase inhibitor) and eicosatetraynoic acid (an arachidonic acid analog that inhibits all arachidonic acid metabolism) severely reduced nodulation in infected insects. The dexamethasone effects were reversed by treating true armyworms with arachidonic acid immediately after infection. In addition to these pharmacological findings, we demonstrate that an eIcosanoid biosynthesis system is present in these insects. Arachidonic acid is present in fat body phospholipids at about 0.4% of total phospholipid fatty acids. Fat body expressed a phospholipase A2 that can hydrolyze arachidonic acid from the sn-2 position of cellular phospholipids. Fat body preparations were competent to biosynthesize prostaglandins, of which PGE2 was the major product. These findings support the hypothesis that eIcosanoids mediate cellular immune reactions in insects.
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EIcosanoids mediate nodulation responses to bacterial infections in larvae of the tenebrionid beetle, Zophobas atratus
Journal of Insect Physiology, 1996Co-Authors: Jon S Miller, Ralph W. Howard, Tuan-duc Nguyen, A. Nguyen, R. M. T. Rosario, David W. Stanley-samuelsonAbstract:Nodulation is the temporally and quantitatively predominant cellular defense response to bacterial infection in insects and other invertebrates. Inhibition of eIcosanoid biosynthesis in larvae of the tenebrionid beetle, Zophobas atratus, immediately prior to intrahemocoelic injections with heat killed preparations of the bacterium, Serratia marcescens, strongly reduced the nodulation response. Separate treatments with specific inhibitors of phospholipase A2, cyclooxygenase, and lipoxygenase reduced nodulation, supporting the view that nodule formation is a complex process involving both cyclooxygenase and lipoxygenase products. The inhibitory effects of the phospholipase A2 inhibitor, dexamethasone, on nodulation were apparent 1 h after infection, and the effects increased, relative to controls, over 24 h. The dexamethasone effects were expressed in a dose-dependent manner, and they were reversed by treating bacteria injected insects with the eIcosanoid-precursor polyunsaturated fatty acid, arachidonic acid (C20:4n-6). Treatments with the saturated fatty acid, 16:0, which is not an eIcosanoid precursor, did not reverse the dexamethasone effects on nodulation. The insects contain low levels of three eIcosanoid precursor polyunsaturated fatty acids in six different tissues, and fat body preparations are competent to produce both cyclooxygenase and lipoxygenase products. These findings strongly support the identification of nodulation as a specific insect cellular defense mechanism that is mediated by eIcosanoids.
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What can we learn from prostaglandins and related eIcosanoids in insects
Insect biochemistry and molecular biology, 1996Co-Authors: David W. Stanley-samuelson, Venkat K PedibhotlaAbstract:Abstract EIcosanoids are oxygenated metabolites of three C20 polyunsaturated fatty acids (20:3n-6, 20:4n-6, and 20:5n-3). While eIcosanoids are very well known in mammalian systems, mostly due to their pharmaceutical interest, there is increasing recognition of the significance of these compounds in insects and other invertebrates. In this paper we consider four major concepts emerging from work on eIcosanoids in invertebrates. First, the biological significance of eIcosanoids extends far beyond their physiological and pathophysiological actions in human and veterinary medicine. Second, we can greatly improve our understanding of eIcosanoids in insects by integrating our work on insects into ongoing studies of other invertebrates. Third, some eIcosanoid actions may be fundamental to animals. Fourth, the biochemistry of eIcosanoids in insects and other invertebrates can differ from expectations based on the mammalian background. Finally, we point to an uncharted frontier in insect studies-the biochemical mechanisms of eIcosanoid action—by drawing attention to some of the work on eIcosanoid receptors in mammalian systems.
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EIcosanoids mediate insect nodulation responses to bacterial infections.
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Jon S Miller, Tuanh Nguyen, David W. Stanley-samuelsonAbstract:We propose that nodule formation is mediated by eIcosanoids in insects. Nodulation is the temporally and quantitatively predominant cellular defense response to bacterial infection in insects and other invertebrates. Inhibition of eIcosanoid biosynthesis in larvae of the tobacco hornworm Manduca sexta immediately prior to intrahemocoelic infections with the bacterium Serratia marcescens strongly reduced the nodulation response. Inhibition of eIcosanoid biosynthesis also reduced formation of cellular aggregates at 1 hr postinfection, which indicates that eIcosanoids mediate early stages of nodulation. Separate treatments with specific inhibitors of phospholipase A2, cyclooxygenase, and lipoxygenase reduced nodulation, which supports the view that nodule formation is a complex process involving prostaglandins and lipoxygenase products. The inhibitory effects of the phospholipase A2 inhibitor dexamethasone on nodulation were apparent by 1 hr after infection, and the effects increased, relative to controls, over 24 hr. The dexamethasone effects were expressed in a dose-dependent manner, and they were reversed by treating infected insects with eIcosanoid-precursor polyunsaturated fatty acids. Treatments with the saturated fatty acid 16:0, which is not an eIcosanoid precursor, did not reverse the dexamethasone effects on nodulation. These findings strongly support the identification of nodulation as a specific insect cellular defense mechanism that is mediated by eIcosanoids.
Alexander A Zoerner - One of the best experts on this subject based on the ideXlab platform.
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analysis of eIcosanoids by lc ms ms and gc ms ms a historical retrospect and a discussion
Journal of Chromatography B, 2014Co-Authors: Dimitrios Tsikas, Alexander A ZoernerAbstract:Abstract EIcosanoids are a large family that derives from arachidonic acid, i.e., eicosatetraenoic acid. Prominent members include prostaglandins, thromboxane and leukotrienes. They are biologically highly active lipid mediators and play multiple physiological roles. GC-MS/MS has played a pivotal role in the identification and quantification of eIcosanoids in biological samples. This technology generated a solid knowledge of their analytical chemistry, biochemistry, physiology and pharmacology. Since about a decade, GC-MS and GC-MS/MS are increasingly displaced by the seemingly more simple, rapid and powerful LC-MS/MS in the area of instrumental analysis of physiological substances, drugs and their metabolites. In this article, we review and discuss LC-MS/MS methods published over the last decade from the perspective of the GC-MS/MS user. Our analysis revealed that the shift from the adult GC-MS/MS to the youthful emerging LC-MS/MS technology in eIcosanoid analysis is associated with several important challenges. Known pitfalls and problematic issues discovered by eIcosanoid pioneers by using GC-MS/MS are often ignored by LC-MS/MS users. Established reference values and intervals provided by GC-MS-based methods are not considered properly in developing and validating LC-MS/MS methods. Virtually, there is a belief in the unlimited capability of the LC-MS/MS technique in eIcosanoid analysis, a thought that simulates analytical certainty. LC-MS/MS users should profit from the plethora of solid knowledge acquired from the use of GC-MS/MS in eIcosanoid analysis in basic and clinical research.
Peter C Isakson - One of the best experts on this subject based on the ideXlab platform.
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rapid quantitation of a large scope of eIcosanoids in two models of inflammation development of an electrospray and tandem mass spectrometry method and application to biological studies
Analytical Biochemistry, 1996Co-Authors: Alon Margalit, Kevin L Duffin, Peter C IsaksonAbstract:Abstract Assessment of eIcosanoid levels in biological systems is important for understanding their role in cell function and pathophysiological events. Current methods of eIcosanoid quantitation are limited by sensitivity, scope, or throughput. The development of a new method for eIcosanoid assessment in biological samples by electrospray and tandem mass spectrometry (MS/MS) in the multiple reaction monitoring mode is described here. In this study, 14 biologically significant eIcosanoids were quantitated in a single sample. Complete sample analysis required two repeated injections of 5 μl with an analysis time of 1.5 min/injection. Limits of detection ranged from 0.5 pg for thromboxane B2(TxB2) to 10 pg for 6-keto prostaglandin F1α(6-keto PGF1α). The reliability, reproducibility, sensitivity, and cross-detection of the method is also described. The MS/MS method was used to explore eIcosanoid production in two inflammation models: lipopolysaccharide (LPS)-stimulated human whole blood and carrageenan-challenged rat air pouch. The most abundant metabolites in LPS-stimulated whole blood were prostaglandin E2(PGE2), TxB2, and 6-keto PGF1α; prostaglandins E1, D2, and F2αand leukotrienes B4and C4were detected in lower amounts. EIcosanoid levels determined by MS/MS were similar to those obtained by immunoassay and GC-MS. The most abundant metabolites detected in carrageenan-challenged rat air pouch were PGE2, 6-keto PGF1α, and TxB2. The method described in this work is accurate and rapid and should greatly aid in evaluating the role of multiple eIcosanoids in future biological studies.
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calcium ionophore a 23187 induced peritoneal eIcosanoid biosynthesis a rapid method to evaluate inhibitors of arachidonic acid metabolism in vivo
Mediators of Inflammation, 1993Co-Authors: Jerry L Currie, Alexander F Shaffer, Peter C IsaksonAbstract:The present investigation characterizes calcium ionophore (A-23187) induced peritoneal eIcosanoid biosynthesis in the rat. Intraperitoneal injection of A-23187 (20 μg/rat) stimulated marked biosynthesis of 6-keto-PGF1α (6-KPA), TxB2, LTC4 and LTB4, with no detectable changes on levels of PGE2. Levels of all eIcosanoids decreased rapidly after a peak which was seen as early as 5 min. Enzyme markers of cellular contents of neutrophils and mononuclear cells, MPO and NAG respectively, decreased rapidly after ionophore injection; this was followed by increases after 60 min. Indomethacin, a selective cyclooxygenase inhibitor, and zileuton and ICI D-2138, two selective 5-lipoxygenase inhibitors attenuated prostaglandin and leukotriene pathways respectively. Oral administration of zileuton (20 mg/kg, p.o.) inhibited LTB4 biosynthesis for up to 6 h suggesting a long duration of pharmacological activity in the rats consistent with its longer half-life. The rapid onset and the magnitude of increases in levels of eIcosanoids render the ionophore induced peritoneal eIcosanoid biosynthesis a useful model to evaluate pharmacological profiles of inhibitors of eIcosanoid pathways in vivo.